The biosynthesis of prothrombin, a glycoprotein with a molecular weight of 72,500, begins with the translation of a liver mRNA for prothrombin precursor which undergoes several post-translational modifications before being secreted into the plasma as mature prothrombin. One of the post-translational modifications, namely the carboxylation of 10 glutamate residues in the N-terminal portion of the molecule to yield gamma carboxy-glutamate, is vitamin K-dependent. Since several proteins with isoelectric points more basic than prothrombin or even acarboxyprothrombin accumulate in the endoplasmic reticulum of the vitamin K deficient rat, it is clear that other modifications of the primordial preprothrombin than carboxylation also occur. A signal sequence may also be present at an early stage. The aims of this research project are to 1) translate the mRNA for preprothrombin in a heterologous cell-free system and determine the amino acid sequence of the product by isotopic micromethods, 2) isolate the 5 isoelectrically distinct preprothrombins (pI equals 7.2, 6.7, 6.2, 5.8 and 5.5) which occur in deficient ER and determine their amino acid sequence, GLA content, extent of glycosylation and other derivatization, 3) determine the physiologic sequence of these prothrombins by pulse-chase experiments in the H-35 hepatoma cell in culture, 4) determine the location of these intermediates in the ER, and 5) to determine the interdependence of CO2 and carbohydrate addition during processing of prothrombin precursor. The attainment of these goals using a line of liver-derived cells in tissue culture and the isolated perfused liver should provide a model for the processing of vitamin K-dependent proteins.